Establishment and Evaluation of Polymerase Chain Reaction for Detection of Y-Chromosome-Specific Fetal DNA in Maternal Blood Circulation during Pregnancy and after Delivery

Abstract

Objective: To establish and evaluate nested polymerase chain reaction (PCR) for detection of Y-chromosome-specific fetal DNA in maternal blood circulation during pregnancy and its clearance several days after delivery. Materials and Methods: Genomic DNA was isolated from a male donor to amplify Y-chromosome-specific DNA sequences by using four pairs of oligonucleotide primers targeting single or multiple copy genes in standard and nested PCR. The nested PCR was evaluated for detection of Y-chromosome-specific sequences in the maternal blood circulation of pregnant women bearing a male fetus during pregnancy and at 2 and 24 h and 7 days after delivery. Results: Although both standard and nested PCR assays were established by using genomic DNA from a male donor to detect Y-chromosome-specific DNA sequences, the nested PCR was 10 times more sensitive than standard PCR. Further experiments showed that nested PCR was able to detect Y-chromosome-specific DNA in the peripheral blood cells as well as in the plasma and serum of pregnant women carrying a male fetus. When tested with plasma samples from women carrying male fetuses during pregnancy, nested PCR was positive with 53/55 (96%), 22/22 (100%), 16/22 (73%) and 0/30 (0%) specimens obtained before and at 2 and 24 h and 7 days after delivery, respectively. These results showed that fetus-specific Y-chromosome DNA was cleared from maternal circulation within 7 days of delivery. Conclusion: Our results suggest that nested PCR is useful in fetal sex determination and that prenatal diagnosis can be done without interference from previous pregnancy.

Polymerase-Chain-Reaction-Based Detection of Fetal Rhesus D and Y-Chromosome-Specific DNA in the Whole Blood of Pregnant Women during Different Trimesters of Pregnancy

“…Primers for the Detection of RhD and Y-Chromosome-Specific DNA Specific primers were used to amplify RhD and Y-chromosome-specific DNA sequences by PCR, as previously reported [9,16] . Following are the designations of the forward (F), reverse (R) and the nested (N) or seminested (SN) primers and their nucleotide sequence.…”

“…The seminested and nested PCRs using the above primers were expected to amplify 118-, 198-and 240-bp DNA fragments from RhD [16] , Y chromosome [9] and ␤ -globin genes [16] , respectively.…”

“…By using the oligonucleotide primers described above, the specific DNA fragments were amplified according to procedures described previously for Y-chromosome-specific DNA [9] . In brief, each reaction mixture (100 l) contained 200 n M of each relevant primer and 2.5 U of AmpliTaq DNA polymerase, PCR buffer, dNTPS and target DNA.…”

“…These methodologies are technically difficult to perform and carry significant risks of complications including pregnancy loss (1-2%) and increased maternal sensitization (40%) [3] . More recently, studies have shown that fetal DNA exists in maternal peripheral circulation [4][5][6][7][8][9] , which led to the establishment of polymerase-chain-reaction (PCR)-based techniques for noninvasive determination of fetal RhD status by using DNA isolated from maternal peripheral blood specimens [10][11][12][13][14][15][16] . These developments represent an important step towards the routine application of noninvasive fetal blood group diagnosis in sensitized pregnancies and may become a model for developing safer noninvasive tests for other single-gene disorders.…”

“…The lower sensitivities of PCR in some of the studies have been attributed to low concentrations of fetal DNA in maternal blood during early stages of pregnancy [20,25] . To further evaluate PCR technique for detection of fetal DNA in maternal blood, in this study we have used highly sensitive seminested and nested PCR assays [9,16] with DNA isolated from whole blood of pregnant women in the first, second and third trimesters of pregnancy to determine the reliability of PCR as a noninvasive technique to determine fetal RhD and gender status during different gestational stages of pregnancy.…”